Module 1 : APPLICATIONS OF PLANT BIOTECHNOLOGY IN CROP IMPROVEMENT
Lecture 13 : Somatic Hybridization and Cybridization
1. Introduction
Sexual hybridization since time immemorial has been used as a method for crop improvement but it has its own limitations as it can only be used within members of same species or closely related wild species. Thus, this limits the use of sexual hybridization as a means of producing better varieties. Development of viable cell hybrids by somatic hybridization, therefore, has been considered as an alternative approach for the production of superior hybrids overcoming the species barrier. The technique can facilitate breeding and gene transfer, bypassing problems associated with conventional sexual crossing such as, interspecific, intergeneric incompatibility. This technique of hybrid production via protoplast fusion allows combining somatic cells (whole or partial) from different cultivars, species or genera resulting in novel genetic combinations including symmetric somatic hybrids, asymmetric somatic hybrids or somatic cybrids.
The most common target using somatic hybridization is the gene of symmetric hybrids that contain the complete nuclear genomes along with cytoplasmic organelles of both parents.
This is unlike sexual reproduction in which organelle genomes are generally contributed by the maternal parent. On the other hand, somatic cybridization is the process of combining the nuclear genome of one parent with the mitochondrial and/or chloroplast genome of a second parent. Cybrids can be produced by donor-recipient method or by cytoplast-protoplast fusion.
Incomplete asymmetric somatic hybridization also provides opportunities for transfer of fragments of the nuclear genome, including one or more intact chromosomes from one parent
(donor) into the intact genome of a second parent (recipient).
These methods provide genetic manipulation of plants overcoming hurdle of sexual incompatibility, thereby, serving as a method of bringing together beneficial traits from taxonomically distinct species which cannot be achieved by sexual crosses. Several parameters such as, source tissue, culture medium and environmental factors influence ability of a protoplast derived hybrid cells to develop into a fertile plant. The general steps involved in somatic hybridization and cybridization methods are elaborated in Figure 13.1 and 13.2 .
Steps involved in somatic hybridization

Figure 13.1: Schematic view of symmetric protoplast fusion producing somatic hybrids.

Figure 13.2: Schematic view of asymmetric protoplast fusion using donor-recipient method
resulting into creation of alloplasmic somatic hybrid or cybrids.
2. Protoplast fusion
Protoplast fusion could be spontaneous during isolation of protoplast or it can be induced by mechanical, chemical and physical means. During spontaneous process, the adjacent protoplasts fuse together as a result of enzymatic degradation of cell walls forming homokaryons or homokaryocytes, each with two to several nuclei. The occurrence of multinucleate fusion bodies is more frequent when the protoplasts are prepared from actively

dividing callus cells or suspension cultures. Since the somatic hybridization or cybridization require fusion of protoplasts of different origin, the spontaneous fusion has no value. To achieve induced fusion, a suitable chemical agent (fusogen) like, NaNO
3
, high Ca 2+ , polyethylene glycol (PEG), or electric stimulus is needed. i. Fusion by means of NaNO
3
: It was first demonstrated by Kuster in 1909 that the hypotonic solution of NaNO
3
induces fusion of isolated protoplast forming heterokaryon
(hybrid). This method was fully described by Evans and Cocking (1975), however this method has a limitation of generating few no of hybrids, especially when highly vacuolated mesophyll protoplasts are involved. ii. High pH and Ca ++ treatment: This technique lead to the development of intra- and interspecific hybrids. It was demonstrated by Keller and Melcher in 1973. The isolated protoplasts from two plant species are incubated in 0.4 M mannitol solution containing high
Ca ++ (50 mM CaCl
2
.2H
2
O) with highly alkaline pH of 10.5 at 37°C for about 30 min.
Aggregation of protoplasts takes place at once and fusion occurs within 10 min. iii. Polyethylene glycol treatment: Polyethylene glycol (PEG) is the most popularly known fusogen due to ability of forming high frequency, binucleate heterokaryons with low cytotoxicity. With PEG the aggregation occurred mostly between two to three protoplasts unlike Ca ++ induced fusion which involves large clump formation. The freshly isolated protoplasts from two selected parents are mixed in appropriate proportions and treated with
15-45% PEG (1500-6000MW) solution for 15-30 min followed by gradual washing of the protoplasts to remove PEG. Protoplast fusion occurs during washing. The washing medium may be alkaline (pH 9-10) and contain a high Ca ++ ion concentration (50 mM). This combined approach of PEG and Ca ++ is much more efficient than the either of the treatment alone. PEG is negatively charged and may bind to cation like Ca ++ , which in turn, may bind to the negatively charged molecules present in plasma lemma, they can also bind to cationic molecules of plasma membrane. During the washing process, PEG molecules may pull out the plasma lemma components bound to them. This would disturb plamalemma organization and may lead to the fusion of protoplasts located close to each other (Figure 13.3). The technique is nonselective thus, induce fusion between any two or more protoplasts.

Figure 13.3: Sequential stages in protoplast fusion. ( A ) two separate protoplasts, ( B ) agglutination of two protoplasts, ( C and D ) Membrane fusion at localized site, and ( E and F ) development of spherical heterokaryon. iv. Electrofusion: The chemical fusion of plant protoplast has many disadvantages – (1) The fusogen are toxic to some cell systems, (2) it produces random, multiple cell aggregates, and
(3) must be removed before culture. Compare to this, electrofusion is rapid, simple, synchronous and more easily controlled. Moreover, the somatic hybrids produced by this method show much higher fertility than those produced by PEG-induced fusion.
Zimmermann and Scheurich (1981) demonstrated that batches of protoplasts could be fused by electric fields by devising a protocol which is now widely used. This protocol involves a two-step process. First, the protoplasts are introduced into a small fusion chamber containing parallel wires or plates which serve as electrodes. Second, a low-voltage and rapidly oscillating AC field is applied, which causes protoplasts to become aligned into chains of cells between electrodes. This creates complete cell-to-cell contact within a few minutes.
Once alignment is complete, the fusion is induced by application of a brief spell of highvoltage DC pulses (o.125-1 kVcm
-1
). A high voltage DC pulses induces a reversible breakdown of the plasma membrane at the site of cell contact, leading to fusion and consequent membrane reorganization. The entire process can be completed within 15 min.
3.
Selection of fusion products
The somatic hybridization by electrofusion of protoplasts allow one-to-one fusion of desired pairs of protoplasts and, therefore, it is easy to know the fate of fusion products. However, protoplast suspension recovered after chemical treatments (fusogen) consists of the following cell types: i. unfused protoplasts of the two species/strains ii. products of fusion between two or more protoplasts of the same species (homokaryons), and iii. ‘hybrid' protoplasts produced by fusion between one (or more) protoplasts of each of the two species (heterokaryons)
The heterokaryons which are the potential source of future hybrids constitute of a very small
(0.5-10%) proportion of the mixture. Therefore, an effective strategy has to be employed for their identification and isolation. Various protocols have been proposed and practiced for the effective selection of hybrids, including morphological basis, complementation of biochemical and genetic traits of the fusing partners, and manual or electronic sorting of heterokaryons/hybrid cells.
3.1. Morpho-physiological basis: The whole mixture of the protoplasts are cultured after fusion treatment and the resulting calli or regenerants are screened for their hybrid characteristics. Occasionally the hybrid calli outgrow the parental cell colonies and are identified by their intermediate morphology, i.e. green with purple coloured cells. However, the process is labour intensive and requires glasshouse facilities. It is limited to certain

combinations showing differences in their regeneration potential under specific culture conditions.
3.2. Complementation: In this case complementation or genetic or metabolic deficiencies of the two fusion partners are utilized to select the hybrid component. When protoplasts of two parents, (one parent bearing cytoplasmic albino trait and the other parent bearing green trait) each parent carrying a non-allelic genetic or metabolic defect are fused, it reconstitutes a viable hybrid cell, of wild type in which both defects are mutually abolished by complementation, and the hybrid cells are able to grow on minimal medium non-permissive to the growth of the parental cells bearing green trait. Later, the calli of hybrid nature could be easily distinguished from the parental type tissue (albino trait) by their green color. The complementation selection can also be applied to dominant characters, such as dominant resistance to antibiotics, herbicides or amino acid analogues.
3.3. Isolation of heterokaryons or hybrid cells: The manual or electronic isolation of heterokaryons or hybrid cells is the most reliable method. Manual isolation requires that the two parental type protoplasts have distinct morphological markers and are easily distinguishable. For example, green vacuolated, mesophyll protoplasts from one parent and richly cytoplasmic, non green protoplasts from cultured cells of another parent. The dual fluorescence method also helps easy identification of fusion products. In this case, the protoplast labeled green by treatment with fluorescein diacetate (FDA, 1-20 mgl
-1
) are fused with protoplasts emitting a red fluorescence, either from chlorophyll autofluorescence or from exogenously applied rhodamine isothiocyanate (10-20 mgl
-1
). The labeling can be achieved by adding the compound into the enzyme mixture. This can be applied even for morphologically indistinguishable protoplasts from two parents.
4. Verification and characterization of somatic hybrids
As no system is foolproof and they have their own advantages and disadvantages. Therefore, even after selecting the desired hybrids/cybrids following protoplast fusion, it is required to carry out one or more tests to compare the parent protoplast lines with the putative hybrids.
Some of the techniques that can be tried are:
4.1. Morphology: Somatic hybrids in most of the cases show characters intermediate between the two parents such as, shape of leaves, pigmentation of corolla, plant height, root morphology and other vegetative and floral characters. The method is not much accurate as tissue culture conditions may also alter some morphological characters or the hybrid may show entirely new traits not shown by any of the parents.
4.2. Isozyme analysis: Multiple molecular forms of same enzyme which catalyses similar or identical reactions are known as isozymes. Electrophoresis is performed to study banding pattern as a check for hybridity. If the two parents exhibit different band patterns for a specific isozyme the putative hybrid can be easily verified. The isozymes commonly used for hybrid identification include, acid phosphatase, esterase, peroxidase.

4.3. Cytological analysis: Chromosome counting of the hybrid is an easier and reliable method to ensure hybridity as it also provides the information of ploidy level. Cytologically the chromosome count of the hybrid should be sum of number of chromosomes from both the parents. Besides number of chromosomes, the size and structure of chromosomes can also be monitored. However, the approach is not applicable to all species, particularly where fusion involves closely related species or where the chromosomes are very small. Moreover, sometimes the somaclonal variations may also give rise to different chromosome number.
4.4. Molecular analysis: Specific restriction pattern of nuclear, mitochondrial and chloroplast
DNA characterizes the plastomes of hybrids and cybrids. Molecular markers such as RFLP,
RAPD, ISSR can be employed to detect variation and similarity in banding pattern of fused protoplasts to verify hybrid and cybrid.
5. Cybrids or cytoplasmic hybrids:
Sexual hybridization involves fusion of the nuclear genes of both the parents but somatic hybrids involves even cytoplasm from both the parental species in hybrid obtained by protoplast fusion. However, in another case somatic hybrids containing nuclear genome of one parent but cytoplasm from both the parents, are termed as cybrids. The approach is time consuming and require several years of crossing plants provides an opportunity to study interparental mitochondrial, chloroplast fusion giving rise to plants with novel genomes.
5.1. Methods to produce cybrids: They are produced in variable frequencies in normal protoplast fusion experiments due to one of the following methods:
1. Fusion of normal protoplast with an enucleated protoplast. The enucleated protoplast can be produced by high speed centrifugation (20,000-40,000xg) for 60 min with 5-50% percoll.
2. Fusion between a normal protoplast and another protoplast with a non-viable nucleus or suppressed nucleus.
3. Elimination of one of the nuclei after heterokaryons formation.
4. Selective elimination of chromosomes at a later stage.
5. Irradiating (with X-rays or gamma rays) the protoplasts of one species prior to fusion in order to inactivate their nuclei.
6. By preparing enucleate protoplasts (cytoplasts) of one species and fusing them with normal protoplasts of the other species.
Cybrids provide the following unique opportunities: (i) transfer of plasmogenes of one species into the nuclear background of another species in a single generation, and even in (ii) sexually incompatible combinations, (iii) recovery of recombinants between the parental mitochondrial or chloroplast DNAs (genomes), and (iv) production of a wide variety of

combinations of the parental and recombinant chloroplasts with the parental or recombinant mitochondria.
6. Applications of somatic hybridization
1. Novel interspecific and intergeneric crosses which are difficult to produce by conventional methods can be easily obtained.
2. Important characters, such as resistance to diseases, ability to undergo abiotic stress and other quality characters, can be obtained in hybrid plant by the fusion of protoplasts of plant bearing particular character to the other plant which may be susceptible to diseases.
3. Protoplasts of sexually sterile haploid, triploid, aneuploid plants can be fused to obtain fertile diploids and polyploids.
4. Studying cytoplasmic genes may be helpful to carry out plant breeding.
5. Most of the agronomically important traits, such as cytoplasmic male sterility, antibiotic resistance and herbicide resistance, are cytoplasmically encoded, hence can be easily transferred to other plant.
6. Plants in juvenile stage can also be hybridized by means of somatic hybridization.
7. Somatic hybridization can be used as a method for the production of autotetraploids.
7. Limitations of somatic hybridization
1. Application of protoplast methodology requires efficient plant regeneration system from isolated protoplasts. Protoplasts from two species can be fused, however, production of somatic hybrids is not easy.
2. Lack of a proper selection method for fused products (hybrids) poses a problem.
3. The end product of somatic hybridization are often unbalanced (sterile, misformed and unstable)
4. Somatic hybridization of two diploids leads to formation of amphidiploids which is unfavorable.
5. It is not sure for a character to completely express after somatic hybridization.
6. The regeneration products of somatic hybridization are often variable due to somaclonal variation, chromosome elimination, organelle segregation.
7. All diverse intergeneric somatic hybrids are sterile and, therefore, have limited chances of development of new varieties.

8. To transfer useful genes from wild species to cultivated crop, it is necessary to achieve intergeneric recombination or chromosome substitution between parental genomes.
Study Questions
1. What are the different methods of protoplast fusion? Explain the mechanism involved in
PEG and High Ca ++ method of fusion.
2. Can you differentiate fusion partners during protoplast fusion?
3. How would you calculate the frequency of heterokaryon formation (Fusion frequency)?
4. Differentiate between the following:
 i. Hybridization and cybridization ii. Homokaryons and heterokaryon iii. Symmetric fusion and asymmetric fusion iv. Spontaneous and induced fusion
Sumber: http://nptel.ac.in/courses/102103016/13